Synthesis and characterization of linear and cyclic
peptoids
-X Cycle- Nuova serie (2008-2011)
Tutor: Prof. Francesco De Riccardis PhD candidate: Chiara De Cola
Co-tutor: Prof. Irene Izzo Coordinatore: Prof. Gaetano Guerra
1
INDEX
CHAPTER 1: INTRODUCTION, 3 1.1 PEPTIDOMIMETICS, 5 1.2 PEPTOIDS: A
PROMISING CLASS OF PEPTIDOMIMETICS, 9 1.3 CONFORMATIONAL STUDIES OF
PEPTOIDS, 11 1.4 PEPTOIDS’ APPLICATIONS, 14 1.5 PEPTOID SINTHESYS,
39 1.6 SYNTHESYS OF PNA MONOMERS AND OLIGOMERS, 41 1.7 AIMS OF THE
WORK, 49 CHAPTER 2: CARBOXYALKYL PEPTOID PNAS: SYNTHESIS AND
HYBRIDIZATION PROPERTIES, 51 2.1 INTRODUCTION, 51 2.2. RESULTS AND
DISCUSSION, 55 2.3. CONCLUSIONS, 60 2.4 EXPERIMENTAL SECTION, 60
CHAPTER 3: STRUCTURAL ANALYSIS OF CYCLOPEPTOIDS AND THEIR
COMPLEXES, 80 3.1 INTRODUCTION, 80 3.2. RESULTS AND DISCUSSION, 85
3.3. CONCLUSIONS, 102 3.4 EXPERIMENTAL SECTION, 103 CHAPTER 4:
CATIONIC CYCLOPEPTOIDS AS POTENTIAL MACROCYCLIC NONVIRAL VECTORS,
115 4.1 INTRODUCTION, 115 4.2. RESULTS AND DISCUSSION, 122 4.3.
CONCLUSIONS, 125 4.4 EXPERIMENTAL SECTION, 125 CHAPTER 5:
COMPLEXATION WITH GD(III) OF CARBOXYETHYL CYCLOPEPTOIDS AS POSSIBLE
CONTRAST AGENTS
IN MRI, 132 5.1 INTRODUCTION, 132 5.2 LARIAT ETHER AND CLICK
CHEMISTRY, 135 5.3 RESULTS AND DISCUSSION, 141 5.4 EXPERIMENTAL
SECTION, 145 CHAPTER 6: CYCLOPEPTOIDS AS MIMETIC OF NATURAL
DEFENSINS, 157 6.1 INTRODUCTION, 157 6.2 RESULTS AND DISCUSSION,
162 6.3 CONCLUSIONS, 167 6.5 EXPERIMENTAL SECTION, 167
2
Chapter 1
1. Introduction
“Giunto a questo punto della vita, quale chimico, davanti alla
tabella del Sistema Periodico, o agli indici
monumentali del Beilstein o del Landolt, non vi ravvisa sparsi i
tristi brandelli, o i trofei, del proprio passato
professionale? Non ha che da sfogliare un qualsiasi trattato, e le
memorie sorgono a grappoli: c’è fra noi chi ha
legato il suo destino, indelebilmente, al bromo o al propilene o al
gruppo –NCO o all’acido glutammico; ed ogni
studente in chimica, davanti ad un qualsiasi trattato, dovrebbe
essere consapevole che in una di quelle pagine, forse in
una sola riga o formula o parola, sta scritto il suo avvenire, in
caratteri indecifrabili, ma che diventeranno chiari
<<POI>>: dopo il successo o l’errore o la colpa, la
vittoria o la disfatta.
Ogni chimico non più giovane, riaprendo alla pagina <<
verhangnisvoll >> quel medesimo trattato, è percosso
da amore o disgusto, si rallegra o si dispera.”.
Da “Il Sistema Periodico”, Primo Levi.
Proteins are vital for essentially every known organism. The
development of a deeper understanding
of protein–protein interactions and the design of novel peptides,
which selectively interact with proteins
are fields of active research.
One way how nature controls the protein functions within living
cells is by regulating protein–
protein interactions. These interactions exist on nearly every
level of cellular function which means they
are of key importance for virtually every process in a living
organism. Regulation of the protein-protein
interactions plays a crucial role in unicellular and multicellular
organisms, including man, and
represents the perfect example of molecular recognition 1 .
Synthetic methods like the solid-phase peptide synthesis (SPPS)
developed by B. Merrifield 2 made it
possible to synthesize polypeptides for pharmacological and
clinical testing as well as for use as drugs
or in diagnostics.
As a result, different new peptide-based drugs are at present
accessible for the treatment of prostate
and breast cancer, as HIV protease inhibitors or as ACE inhibitors
to treat hypertension and congestive
heart failures, to mention only few examples 1 .
Unfortunately, these small peptides typically show high
conformational flexibility and a low in-vivo
stability which hampers their application as tools in medicinal
diagnostics or molecular biology. A
major difficulty in these studies is the conformational flexibility
of most peptides and the high
dependence of their conformations on the surrounding environment
which often leads to a
conformational equilibrium. The high flexibility of natural
polypeptides is due to the multiple
conformations that are energetically possible for each residue of
the incorporated amino acids. Every
amino acid has two degrees of conformational freedom, N–Cα (Φ) and
Cα–CO (Ψ) resulting in
approximately 9 stable local conformations 1 . For a small peptide
with only 40 amino acids in length the
1 A. Grauer, B. König Eur. J. Org. Chem. 2009, 5099–5111.
2 a) R. B. Merrifield, Federation Proc. 1962, 21, 412; b) R. B.
Merrifield, J. Am. Chem. Soc. 1964, 86, 2149–2154.
4
number of possible conformations which need to be considered
escalates to nearly 1040 3 . This
extraordinary high flexibility of natural amino acids leads to the
fact that short polypeptides consisting
of the 20 proteinogenic amino acids rarely form any stable 3D
structures in solution 1 . There are only
few examples reported in the literature where short to medium-sized
peptides (<30–50 amino acids)
were able to form stable structures. In most cases they exist in
aqueous solution in numerous
dynamically interconverting conformations. Moreover, the number of
stable short peptide structures,
which are available is very limited, because of the need to use
amino acids having a strong structure
inducing effect like for example helix-inducing amino acids as
leucine, glutamic acid or lysine. In
addition, it is dubious whether the solid state conformations
determined by X-ray analysis are identical
to those occurring in solution or during the interactions of
proteins with each other 1 . Despite their wide
range of important bioactivities, polypeptides are generally poor
drugs. Typically, they are rapidly
degraded by proteases in vivo, and are frequently
immunogenic.
This fact has inspired prevalent efforts to develop peptide mimics
for biomedical applications, a task
that presents formidable challenges in molecular design.
1.1 Peptidomimetics
One very versatile strategy to overcome such drawbacks is the use
of peptidomimetics 4 . These are
small molecules, which mimic natural peptides or proteins and thus
produce the same biological effects
as their natural role models.
They also often show a decreased activity in comparison to the
protein from which they are derived.
These mimetics should have the ability to bind to their natural
targets in the same way as the natural
peptide sequences, from which their structure was derived, do and
should produce the same biological
effects. It is possible to design these molecules in such a way
that they show the same biological effects
as their peptide role models but with enhanced properties like a
higher proteolytic stability, higher
bioavailability and also often with improved selectivity or
potency. This makes them interesting targets
for the discovery of new drug candidates.
For the progress of potent peptidomimetics, it is required to
understand the forces that lead to
protein–protein interactions with nanomolar or often even higher
affinities.
These strong interactions between peptides and their corresponding
proteins are mainly based on side
chain interactions indicating that the peptide backbone itself is
not an absolute requirement for high
affinities.
This allows chemists to design peptidomimetics basically from any
scaffold known in chemistry by
replacing the amide backbone partially or completely by other
structures. Peptidomimetics, furthermore,
can have some peculiar qualities, such as a good solubility in
aqueous solutions, access to facile
sequences-specific assembly of monomers containing chemically
diverse side chains and the capacity to
form stable, biomimetic folded structures 5 .
Most important is that the backbone is able to place the amino acid
side chains in a defined 3D-
position to allow interactions with the target protein, too.
Therefore, it is necessary to develop an idea of
the required structure of the peptidomimetic to show a high
activity against its biological target.
3 J. Venkatraman, S. C. Shankaramma, P. Balaram, Chem. Rev. 2001,
101, 3131–3152. 4 J. A. Patch, K. Kirshenbaum, S. L. Seurynck, R.
N. Zuckermann and A. E. Barron, in Pseudo-peptides in Drug
Development, ed. P. E. Nielsen, Wiley-VCH, Weinheim, Germany, 2004,
1–31.
5
The most significant parameters for an optimal peptidomimetics are:
stereochemistry, charge and
hydrophobicity, and these parameters can be examined by systematic
exchange of single amino acids
with modified amino acid. As a result, the key residues, which are
essential for the biological activity,
can be identified. As next step the 3D arrangement of these key
residues needs to be analyzed by the use
of compounds with rigid conformations to identify the most active
structure 1 . In general, the
development of peptidomimetics is based mainly on the knowledge of
the electronic, conformational
and topochemical properties of the native peptide to its
target.
Two structural factors are particularly important for the synthesis
of peptidomimetics with high
biological activity: firstly the mimetic has to have a convenient
fit to the binding site and secondly the
functional groups, polar and hydrophobic regions of the mimetic
need to be placed in defined positions
to allow the useful interactions to take place 1 .
One very successful approach to overcome these drawbacks is the
introduction of conformational
constraints into the peptide sequence. This can be done for example
by the incorporation of amino acids,
which can only adopt a very limited number of different
conformations, or by cyclisation (main chain to
main chain; side chain to main chain or side chain to side chain).
5
Peptidomimetics, furthermore, can contain two different
modifications: amino acid modifications or
peptides‘ backbone modifications.
Figure 1.1 reports the most important ways to modify the backbone
of peptides at different positions.
Figure 1.1. Some of the more common modifications to the peptide
backbone (adapted from
literature). 6
5a) C. Toniolo, M. Goodman, Introduction to the Synthesis of
Peptidomimetics, in: Methods of Organic Chemistry:
Synthesis of Peptides and Peptidomimetics (Ed.: M. Goodman),
Thieme, Stuttgart, New York, 2003, vol. E22c, p.
1–2; b) D. J. Hill, M. J. Mio, R. B. Prince, T. S. Hughes, J. S.
Moore, Chem. Rev. 2001, 101, 3893–4012. 6 J. Gante, Angew. Chem.
Int. Ed. Engl. 1994, 33, 1699–1720.
6
is possible:
the replacement of the α-CH group by nitrogen to form
azapeptides,
the change from amide to ester bond to get depsipeptides,
the exchange of the carbonyl function by a CH2 group,
the extension of the backbone (β-amino acids and γ-amino
acids),
the amide bond inversion (a retro-inverse peptidomimetic),
The carba, alkene or hydroxyethylene groups are used in exchange
for the amide bond.
The shift of the alkyl group from α-CH group to α-N group.
Most of these modifications do not guide to a higher restriction of
the global conformations, but they
have influence on the secondary structure due to the altered
intramolecular interactions like different
hydrogen bonding. Additionally, the length of the backbone can be
different and a higher proteolytic
stability occurs in most cases 1 .
1.2 Peptoids: A Promising Class of Peptidomimetics.
If we shift the chain of α-CH group by one position on the peptide
backbone, we produced the
disappearance of all the intra-chain stereogenic centers and the
formation of a sequence of variously
substituted N-alkylglycines (figure 1.2).
Figure 1.2. Comparison of a portion of a peptide chain with a
portion of a peptoid chain.
Oligomers of N-substituted glycine, or peptoids, were developed by
Zuckermann and co-workers in
the early 1990‘s 7 . They were initially proposed as an accessible
class of molecules from which lead
compounds could be identified for drug discovery.
Peptoids can be described as mimics of α-peptides in which the side
chain is attached to the
backbone amide nitrogen instead of the α-carbon (figure 1.2). These
oligomers are an attractive scaffold
for biological applications because they can be generated using a
straightforward, modular synthesis that
allows the incorporation of a wide variety of functionalities 8 .
Peptoids have been evaluated as tools to
7 R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A.
Jewell, S. Banville, S. Ng, L.Wang, S.
Rosenberg, C. K. Marlowe, D. C. Spellmeyer, R. Tan, A. D. Frankel,
D. V. Santi, F. E. Cohen and P. A. Bartlett,
Proc. Natl. Acad. Sci. U. S. A., 1992, 89, 9367–9371.
7
study biomolecular interactions, 8 and also hold significant
promise for therapeutic applications due to
their enhanced proteolytic stabilities 8 and increased cellular
permeabilities
9 relative to α-peptides.
Biologically active peptoids have also been discovered by rational
design (i.e., using molecular
modeling), and were synthesized either individually or in parallel
focused libraries 10
. For some
applications, a well-defined structure is also necessary for
peptoid function to display the functionality
in a particular orientation, or to adopt a conformation that
promotes interaction with other molecules.
However, in other biological applications, peptoids lacking defined
structures appear to possess superior
activities over structured peptoids.
This introduction will focus primarily on the relationship between
peptoid structure and function. A
comprehensive review of peptoids in drug discovery, detailing
peptoid synthesis, biological
applications, and structural studies, was published by Barron,
Kirshenbaum, Zuckermann, and co-
workers in 2004 4 . Since then, significant advances have been made
in these areas, and new applications
for peptoids have emerged. In addition, new peptoid secondary
structural motifs have been reported, as
well as strategies to stabilize those structures. Lastly, the
emergence of peptoid with tertiary structures
has driven chemists towards new structures with peculiar properties
and side chains. Peptoid monomers
are linked through polyimide bonds, in contrast to the amide bonds
of peptides. Unfortunately, peptoids
do not have the hydrogen of the peptide secondary amide, and are
consequently incapable of forming
the same types of hydrogen bond networks that stabilize peptide
helices and β-sheets.
The peptoids oligomers backbone is achiral; however stereogenic
centers can be included in the side
chains to obtain secondary structures with a preferred handedness 4
. In addition, peptoids carrying N-
substituted versions of the proteinogenic side chains are highly
resistant to degradation by proteases,
which is an important attribute of a pharmacologically useful
peptide mimic 4 .
1.3 Conformational studies of peptoids
The fact that peptoids are able to form a variety of secondary
structural elements, including helices
and hairpin turns, suggests a range of possible conformations that
can allow the generation of functional
folds. 11
Some studies of molecular mechanics, have demonstrated that peptoid
oligomers bearing bulky
chiral (S)-N-(1-phenylethyl) side chains would adopt a polyproline
type I helical conformation, in
agreement with subsequent experimental findings 12
.
Kirshenbaum at al.12 has shown agreement between theoretical models
and the trans amide of N-
aryl peptoids, and suggested that they may form polyproline type II
helices. Combined, these studies
suggest that the backbone conformational propensities evident at
the local level may be readily
translated into the conformations of larger oligomers chains.
N-α-chiral side chains were shown to promote the folding of these
structures in both solution and the
solid state, despite the lack of main chain chirality and secondary
amide hydrogen bond donors crucial
to the formation of many α-peptide secondary structures.
8 S. M. Miller, R. J. Simon, S. Ng, R. N. Zuckermann, J. M. Kerr,
W. H. Moos, Bioorg. Med. Chem. Lett., 1994, 4,
2657–2662. 9 Y. U.Kwon and T. Kodadek, J. Am. Chem. Soc., 2007,
129, 1508–1509. 10 T. Hara, S. R. Durell, M. C. Myers and D. H.
Appella, J. Am. Chem. Soc., 2006, 128, 1995–2004. 11 G. L.
Butterfoss, P. D. Renfrew, B. Kuhlman, K. Kirshenbaum, R. Bonneau,
J. Am. Chem. Soc., 2009, 131,
16798–16807.
While computational studies initially suggested that steric
interactions between N-α-chiral aromatic
side chains and the peptoid backbone primarily dictated helix
formation, both intra- and intermolecular
aromatic stacking interactions 12
.
selected side chain functionalities to look at the
effects of four key types of noncovalent interactions on peptoid
amide cis/trans equilibrium: (1) n→π*
interactions between an amide and an aromatic ring (n→π*Ar), (2)
n→π* interactions between two
carbonyls (n→π* C=O), (3) side chain-backbone steric interactions,
and (4) side chain-backbone
hydrogen bonding interactions. In figure 1.3 are reported, as
example, only n→π*Ar and n→π*C=O
interactions.
A B
Figure 1.3. A: (Left) n→π*Ar interaction (indicated by the red
arrow) proposed to increase of
Kcis/trans (equilibrium constant between cis and trans
conformation) for peptoid backbone amides. (Right)
Newman projection depicting the n→π*Ar interaction. B: (Left)
n→π*C=O interaction (indicated by
the red arrow) proposed to reduce Kcis/trans for the donating amide
in peptoids. (Right) Newman
projection depicting the n→π*C=O interaction.
Other classes of peptoid side chains have been designed to
introduce dipole-dipole, hydrogen
bonding, and electrostatic interactions stabilizing the peptoid
helix.
In addition, such constraints may further rigidify peptoid
structure, potentially increasing the ability
of peptoid sequences for selective molecular recognition.
In a relatively recent contribution Kirshenbaum 15
reported that peptoids undergo to a very efficient
head-to-tail cyclisation using standard coupling agents. The
introduction of the covalent constraint
enforces conformational ordering, thus facilitating the
crystallization of a cyclic peptoid hexamer and a
cyclic peptoid octamer.
Peptoids can form well-defined three-dimensional folds in solution,
too. In fact, peptoid oligomers
with α-chiral side chains were shown to adopt helical structures
16
; a threaded loop structure was formed
12 C. W. Wu, T. J. Sanborn, R. N. Zuckermann, A. E. Barron, J. Am.
Chem. Soc. 2001, 123, 2958–2963. 13 T. J. Sanborn, C. W. Wu, R. N.
Zuckermann, A. E. Barron, Biopolymers, 2002, 63, 12–20. 14
B. C. Gorske, J. R. Stringer, B. L. Bastian, S. A. Fowler, H. E.
Blackwell , J. Am. Chem. Soc., 2009, 131,
16555–16567. 15 S. B. Y. Shin, B. Yoo, L. J. Todaro, K.
Kirshenbaum, J. Am. Chem. Soc., 2007, 129, 3218-3225. 16 (a) K.
Kirshenbaum, A. E. Barron, R. A. Goldsmith, P. Armand, E. K.
Bradley, K. T. V. Truong, K. A. Dill, F. E.
Cohen, R. N. Zuckermann, Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
4303–4308. (b) P. Armand, K. Kirshenbaum, R.
A. Goldsmith, S. Farr-Jones, A. E. Barron, K. T. V. Truong, K. A.
Dill, D. F. Mierke, F. E. Cohen, R. N.
Zuckermann, E. K. Bradley, Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
4309–4314. (c) C. W. Wu, K. Kirshenbaum, T.
J. Sanborn, J. A. Patch, K. Huang, K. A. Dill, R. N. Zuckermann, A.
E. Barron, J. Am. Chem. Soc. 2003, 125,
13525–13530.
; head-to-tail macrocyclizations provided
conformationally restricted cyclic peptoids.
These studies demonstrate the importance of (1) access to
chemically diverse monomer units and (2)
precise control of secondary structures to expand applications of
peptoid helices.
The degree of helical structure increases as chain length grows,
and for these oligomers becomes
fully developed at length of approximately 13 residues. Aromatic
side chain-containing peptoid helices
generally give rise to CD spectra that are strongly reminiscent of
that of a peptide α-helix, while peptoid
helices based on aliphatic groups give rise to a CD spectrum that
resembles the polyproline type-I
helical.
The well-defined helical structure associated with appropriately
substituted peptoid oligomers can be
employed to construct compounds that closely mimic the structures
and functions of certain bioactive
peptides. In this paragraph, are shown some examples of peptoids
that have antibacterial and
antimicrobial properties, molecular recognition properties, of
metal complexing peptoids, of catalytic
peptoids, and of peptoids tagged with nucleobases.
1.4.1 Antibacterial and antimicrobial properties
The antibiotic activities of structurally diverse sets of
peptides/peptoids derive from their action on
microbial cytoplasmic membranes. The model proposed by
Shai–Matsuzaki–Huan 17
(SMH) presumes
membrane functions. Cyclization of linear peptide/peptoid
precursors (as a mean to obtain
conformational order), has been often neglected 18
, despite the fact that nature offers a vast assortment of
powerful cyclic antimicrobial peptides 19
. However, macrocyclization of N-substituted glycines gives
17 (a) Matsuzaki, K. Biochim. Biophys. Acta 1999, 1462, 1; (b)
Yang, L.; Weiss, T. M.; Lehrer, R. I.; Huang, H. W.
Biophys. J. 2000, 79, 2002; (c) Shai, Y. Biochim.Biophys. Acta
1999, 1462, 55. 18 Chongsiriwatana, N. P.; Patch, J. A.; Czyzewski,
A. M.; Dohm, M. T.; Ivankin, A.; Gidalevitz, D.; Zuckermann,
R. N.; Barron, A. E. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2794.
19 Interesting examples are: (a) Motiei, L.; Rahimipour, S.;
Thayer, D. A.; Wong, C. H.; Ghadiri, M. R. Chem.
Commun. 2009, 3693; (b) Fletcher, J. T.; Finlay, J. A.; Callow, J.
A.; Ghadiri, M. R. Chem. Eur. J. 2007, 13, 4008;
(c) Au, V. S.; Bremner, J. B.; Coates, J.; Keller, P. A.; Pyne, S.
G. Tetrahedron 2006, 62, 9373; (d) Fernandez-
Lopez, S.; Kim, H.-S.; Choi, E. C.; Delgado, M.; Granja, J. R.;
Khasanov, A.; Kraehenbuehl, K.; Long, G.;
Weinberger, D. A.; Wilcoxen, K. M.; Ghadiri, M. R. Nature 2001,
412, 452; (e) Casnati, A.; Fabbi, M.; Pellizzi, N.;
Pochini, A.; Sansone, F.; Ungaro, R.; Di Modugno, E.; Tarzia, G.
Bioorg. Med. Chem. Lett. 1996, 6, 2699; (f)
Robinson, J. A.; Shankaramma, C. S.; Jetter, P.; Kienzl, U.;
Schwendener, R. A.; Vrijbloed, J. W.; Obrecht, D.
Bioorg. Med. Chem. 2005, 13, 2055.
10
and excellent membrane-permeabilizing
.
Antimicrobial peptides (AMPs) are found in myriad organisms and are
highly effective against
bacterial infections 23
. The mechanism of action for most AMPs is permeabilization of the
bacterial
.
The cationic region of AMPs confers a degree of selectivity for the
membranes of bacterial cells over
mammalian cells, which have negatively charged and neutral
membranes, respectively. The
hydrophobic portions of AMPs are supposed to mediate insertion into
the bacterial cell membrane.
Although AMPs possess many positive attributes, they have not been
developed as drugs due to the
poor pharmacokinetics of α-peptides. This problem creates an
opportunity to develop peptoid mimics of
.
De Riccardis 26
et al. investigated the antimicrobial activities of five new cyclic
cationic hexameric α-
peptoids comparing their efficacy with the linear cationic and the
cyclic neutral counterparts (figure
1.4).
20 (a) Craik, D. J.; Cemazar, M.; Daly, N. L. Curr. Opin. Drug
Discovery Dev. 2007, 10, 176; (b) Trabi, M.; Craik,
D. J. Trend Biochem. Sci. 2002, 27, 132. 21 (a) Maulucci, N.; Izzo,
I.; Bifulco, G.; Aliberti, A.; De Cola, C.; Comegna, D.; Gaeta, C.;
Napolitano, A.; Pizza,
C.; Tedesco, C.; Flot, D.; De Riccardis, F. Chem. Commun. 2008,
3927; (b) Kwon, Y.-U.; Kodadek, T. Chem.
Commun. 2008, 5704; (c) Vercillo, O. E.; Andrade, C. K. Z.;
Wessjohann, L. A. Org. Lett. 2008, 10, 205; (d) Vaz,
B.; Brunsveld, L. Org. Biomol. Chem. 2008, 6, 2988; (e) Wessjohann,
L. A.; Andrade, C. K. Z.; Vercillo, O. E.;
Rivera, D. G.. In Targets in Heterocyclic Systems; Attanasi, O. A.,
Spinelli, D., Eds.; Italian Society of Chemistry,
2007; Vol. 10, pp 24–53; (f) Shin, S. B. Y.; Yoo, B.; Todaro, L.
J.; Kirshenbaum, K. J. Am. Chem. Soc. 2007, 129,
3218; (g) Hioki, H.; Kinami, H.; Yoshida, A.; Kojima, A.; Kodama,
M.; Taraoka, S.; Ueda, K.; Katsu, T.
Tetrahedron Lett. 2004, 45, 1091. 22 (a) Chatterjee, J.; Mierke,
D.; Kessler, H. Chem. Eur. J. 2008, 14, 1508; (b) Chatterjee, J.;
Mierke, D.; Kessler,
H. J. Am. Chem. Soc. 2006, 128, 15164; (c) Nnanabu, E.; Burgess, K.
Org. Lett. 2006, 8, 1259; (d) Sutton, P. W.;
Bradley, A.; Farràs, J.; Romea, P.; Urpì, F.; Vilarrasa, J.
Tetrahedron 2000, 56, 7947; (e) Sutton, P. W.; Bradley,
A.; Elsegood, M. R.; Farràs, J.; Jackson, R. F. W.; Romea, P.;
Urpì, F.; Vilarrasa, J. Tetrahedron Lett. 1999, 40,
2629. 23 A. Peschel and H.-G. Sahl, Nat. Rev. Microbiol., 2006, 4,
529–536. 24 R. E. W. Hancock and H.-G. Sahl, Nat. Biotechnol.,
2006, 24, 1551–1557. 25 For a review of antimicrobial peptoids,
see: I. Masip, E. Pèrez Payà, A. Messeguer, Comb. Chem. High
Throughput Screen., 2005, 8, 235–239. 26 D. Comegna, M. Benincasa,
R. Gennaro, I. Izzo, F. De Riccardis, Bioorg. Med. Chem,. 2010, 18,
2010–2018.
11
Figure 1.4. Structures of synthesized linear and cyclic peptoids
described by De Riccardis at al. Bn
= benzyl group; Boc= t-butoxycarbonyl group.
The synthesized peptoids have been assayed against clinically
relevant bacteria and fungi, including
Escherichia coli, Staphylococcus aureus, amphotericin β-resistant
Candida albicans, and Cryptococcus
neoformans 27
.
The purpose of this study was to explore the biological effects of
the cyclisation on positively
charged oligomeric N-alkylglycines, with the idea to mimic the
natural amphiphilic peptide antibiotics.
The long-term aim of the effort was to find a key for the rational
design of novel antimicrobial
compounds using the finely tunable peptoid backbone.
The exploration for possible biological activities of linear and
cyclic α-peptoids, was started with the
assessment of the antimicrobial activity of the known 21a
N-benzyloxyethyl cyclohomohexamer (Figure
1.4, Block I). This neutral cyclic peptoid was considered a
promising candidate in the antimicrobial
27 M. Benincasa, M. Scocchi, S. Pacor, A. Tossi, D. Nobili, G.
Basaglia, M. Busetti, R. J. Gennaro, Antimicrob.
Chemother. 2006, 58, 950.
12
assays for its high affinity to the first group alkali metals (Ka ~
106 for Na + , Li
+ and K
28 , a behavior similar to that
observed for valinomycin, a well known K + -carrier with powerful
antibiotic activity
29 . However,
determination of the MIC values showed that neutral chains did not
exert any antimicrobial activity
against a group of selected pathogenic fungi, and of Gram-negative
and Gram-positive bacterial strains
even at concentrations up to 1 mM.
Detailed structure–activity relationship (SAR) studies 30
have revealed that the amphiphilicity of the
peptides/peptidomimetics and the total number of positively charged
residues, impact significantly on
the antimicrobial activity. Therefore, cationic versions of the
neutral cyclic α-peptoids were planned
(Figure 1.4, block I and block II compounds). In this study were
also included the linear cationic
precursors to evaluate the effect of macrocyclization on the
antimicrobial activity. Cationic peptoids
were tested against four pathogenic fungi and three clinically
relevant bacterial strains. The tests showed
a marked increase of the antibacterial and antifungal activities
with cyclization. The presence of charged
amino groups also influenced the antimicrobial efficacy, as shown
by the activity of the bi- and
tricationic compounds, when compared with the ineffective neutral
peptoid. These results are the first
indication that cyclic peptoids can represent new motifs on which
to base artificial antibiotics.
In 2003, Barron and Patch 31
reported peptoid mimics of the helical antimicrobial peptide
magainin-2
that had low micromolar activity against Escherichia coli (MIC =
5–20 mM) and Bacillus subtilis (MIC
= 1–5 mM).
The magainins exhibit highly selective and potent antimicrobial
activity against a broad spectrum of
organisms 5 . As these peptides are facially amphipathic, the
magainins have a cationic helical face
mostly composed of lysine residues, as well as hydrophobic aromatic
(phenylalanine) and hydrophobic
aliphatic (valine, leucine and isoleucine) helical faces. This
structure is responsible for their activity 4 .
Peptoids have been shown to form remarkably stable helices, with
physical characteristics similar to
those of peptide polyproline type-I helices. In fact, a series of
peptoid magainin mimics with this type
of three-residue periodic sequences has been synthesized 4 and
tested against E. coli JM109 and B.
subtulis BR151. In all cases, peptoids are individually more active
against the Gram-positive species.
The amount of hemolysis induced by these peptoids correlated well
with their hydrophobicity. In
summary, these recently obtain results demonstrate that certain
amphipathic peptoid sequences are also
capable of antibacterial activity.
1.4.2 Molecular Recognition
Peptoids are currently being studied for their potential to serve
as pharmaceutical agents and as
chemical tools to study complex biomolecular interactions.
Peptoid–protein interactions were first
demonstrated in a 1994 report by Zuckermann and co-workers, 8 where
the authors examined the high-
affinity binding of peptoid dimers and trimers to G-protein-coupled
receptors. These groundbreaking
studies have led to the identification of several peptoids with
moderate to good affinity and, more
28 C. De Cola, S. Licen, D. Comegna, E. Cafaro, G. Bifulco, I.
Izzo, P. Tecilla, F. De Riccardis, Org. Biomol.
Chem. 2009, 7, 2851. 29 N. R. Clement, J. M. Gould, Biochemistry,
1981, 20, 1539. 30 J. I. Kourie, A. A. Shorthouse, Am. J. Physiol.
Cell. Physiol. 2000, 278, C1063. 31 J. A. Patch and A. E. Barron,
J. Am. Chem. Soc., 2003, 125, 12092– 12093.
13
importantly, excellent selectivity for protein targets that
implicated in a range of human diseases. There
are many different interactions between peptoid and protein, and
these interactions can induce a certain
inhibition, cellular uptake and delivery. Synthetic molecules
capable of activating the expression of
specific genes would be valuable for the study of biological
phenomena and could be therapeutically
useful. From a library of ~100000 peptoid hexamers, Kodadek and
co-workers recently identified three
peptoids (24-26) with low micromolar binding affinities for the
coactivator CREB-binding protein
(CBP) in vitro (Figure 1.5) 9 . This coactivator protein is
involved in the transcription of a large number
of mammalian genes, and served as a target for the isolation of
peptoid activation domain mimics. Of
the three peptoids, only 24 was selective for CBP, while peptoids
25 and 26 showed higher affinities for
bovine serum albumin. The authors concluded that the promiscuous
binding of 25 and 26 could be
attributed to their relatively sticky natures (i.e., aromatic,
hydrophobic amide side chains).
Inhibitors of proteasome function that can intercept proteins
targeted for degradation would be
valuable as both research tools and therapeutic agents. In 2007,
Kodadek and co-workers 32
identified the
first chemical modulator of the proteasome 19S regulatory particle
(which is part of the 26S proteasome,
an approximately 2.5 MDa multi-catalytic protease complex
responsible for most non-lysosomal protein
degradation in eukaryotic cells). A one bead one compound peptoid
library was constructed by split
and pool synthesis.
Figure 1.5. Peptoid hexamers 24, 25, and 26 reported by Kodadek and
co-workers and their
dissociation constants (KD) for coactivator CBP 33
. Peptoid 24 was able to function as a transcriptional
activation domain mimic (EC50 = 8 mM).
32 H. S. Lim, C. T. Archer, T. Kodadek J. Am. Chem. Soc., 2007,
129, 7750.
14
Each peptoid molecule was capped with a purine analogue in hope of
biasing the library toward
targeting one of the ATPases, which are part of the 19S regulatory
particle. Approximately 100 000
beads were used in the screen and a purine-capped peptoid heptamer
(27, Figure 1.6) was identified as
the first chemical modulator of the 19S regulatory particle. In an
effort to evidence the pharmacophore
of 27 33
(by performing a glycine scan, similar to the alanine scan in
peptides) it was shown that just
the core tetrapeptoid was necessary for the activity.
Interestingly, the synthesis of the shorter peptoid 27 gave, in the
experiments made on cells, a 3- to
5-fold increase in activity relative to 28. The higher activity in
the cell-based essay was likely due to
increased cellular uptake, as 27 does not contain charged
residues.
Figure 1.6. Purine capped peptoid heptamer (28) and tetramer (27)
reported by Kodadek preventing
protein degradation
1.4.3 Metal Complexing Peptoids
A desirable attribute for biomimetic peptoids is the ability to
show binding towards receptor sites.
This property can be evoked by proper backbone folding due
to:
1) local side-chain stereoelectronic influences,
2) coordination with metallic species,
3) presence of hydrogen-bond donor/acceptor patterns.
Those three factors can strongly influence the peptoids‘ secondary
structure, which is difficult to
observe due to the lack of the intra-chain C=OH–N bonds, present in
the parent peptides.
Most peptoids‘ activities derive by relatively unstructured
oligomers. If we want to mimic the
sophisticated functions of proteins, we need to be able to form
defined peptoid tertiary structure folds
and introduce functional side chains at defined locations. Peptoid
oligomers can be already folded into
helical secondary structures. They can be readily generated by
incorporating bulky chiral side chains
33 H.S. Lim, C. T. Archer, Y. C. Kim, T. Hutchens, T. Kodadek Chem.
Commun., 2008, 1064.
15
. Such helical secondary structures are extremely stable to
chemical denaturants
and temperature 13
. The unusual stability of the helical structure may be a
consequence of the steric
.
Zuckermann and co-workers synthesized biomimetic peptoids with
zinc-binding sites 8 , since zinc-
binding motifs in protein are well known. Zinc typically stabilizes
native protein structures or acts as a
cofactor for enzyme catalysis 37-38
. Zinc also binds to cellular cysteine-rich metallothioneins solely
for
storage and distribution 39
. The binding of zinc is typically mediated by cysteines and
histidines
50-51 . In
order to create a zinc-binding site, they incorporated thiol and
imidazole side chains into a peptoid two-
helix bundle.
Classic zinc-binding motifs, present in proteins and including
thiol and imidazole moieties, were
aligned in two helical peptoid sequences, in a way that they could
form a binding site. Fluorescence
resonance energy transfer (FRET) reporter groups were located at
the edge of this biomimetic structure
in order to measure the distance between the two helical segments
and probe and, at the same time, the
zinc binding propensity (29, Figure 1.7).
29
Figure 1.7. Chemical structure of 29, one of the twelve folded
peptoids synthesized by Zuckermann,
able to form a Zn 2+
complex.
Folding of the two helix bundles was allowed by a Gly-Gly-Pro-Gly
middle region. The study
demonstrated that certain peptoids were selective zinc binders at
nanomolar concentration.
The formation of the tertiary structure in these peptoids is
governed by the docking of preorganized
peptoid helices as shown in these studies 40
.
A survey of the structurally diverse ionophores demonstrated that
the cyclic arrangement represents a
common archetype equally promoted by chemical design 22f
and evolutionary pressure. Stereoelectronic
peptoids‘ achiral polyimide backbone. In particular, the prediction
and the assessment of the covalent
34 Wu, C. W.; Kirshenbaum, K.; Sanborn, T. J.; Patch, J. A.; Huang,
K.; Dill, K. A.; Zuckermann, R. N.; Barron, A.
E. J. Am. Chem. Soc. 2003, 125, 13525–13530. 35 Armand, P.;
Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L., Jr.; Dill,K. A.;
Zuckermann, R. N.; Cohen, F. E.
Folding Des. 1997, 2, 369–375. 36 K. Kirshenbaum, R. N. Zuckermann,
K. A. Dill, Curr. Opin. Struct. Biol. 1999, 9, 530–535. 37 Coleman,
J. E. Annu. ReV. Biochem. 1992, 61, 897–946. 38 Berg, J. M.;
Godwin, H. A. Annu. ReV. Biophys. Biomol. Struct. 1997, 26,
357–371. 39 Cousins, R. J.; Liuzzi, J. P.; Lichten, L. A. J. Biol.
Chem. 2006, 281, 24085–24089. 40 B. C. Lee, R. N. Zuckermann, K. A.
Dill, J. Am. Chem. Soc. 2005, 127, 10999–11009.
16
constraints induced by macrolactamization appears crucial for the
design of conformationally restricted
peptoid templates as preorganized synthetic scaffolds or receptors.
In 2008 were reported the synthesis
and the conformational features of cyclic tri-, tetra-, hexa-, octa
and deca- N-benzyloxyethyl glycines
(30-34, figure 1.8) 21a
.
Figure 1.8. Structure of cyclic tri-, tetra-, hexa-, octa and deca-
N-benzyloxyethyl glycines.
It was found, for the flexible eighteen-membered N-benzyloxyethyl
cyclic peptoid 32, high binding
constants with the first group alkali metals (Ka ~ 106 for Na + ,
Li
+ and K
+ ), while, for the rigid cis–
trans–cis–trans cyclic tetrapeptoid 31, there was no evidence of
alkali metals complexation. The
conformational disorder in solution was seen as a propitious
auspice for the complexation studies. In
fact, the stepwise addition of sodium picrate to 32, induced the
formation of a new chemical species,
whose concentration increased with the gradual addition of the
guest. The conformational equilibrium
between the free host and the sodium complex, resulted in being
slower than the NMR-time scale,
giving, with an excess of guest, a remarkably simplified 1 H NMR
spectrum, reflecting the formation of
a 6-fold symmetric species (Figure 1.9).
Figure 1.9. Picture of the predicted lowest energy conformation for
the complex 32 with sodium.
A conformational search on 32 as a sodium complex suggested the
presence of an S6-symmetry axis
passing through the intracavity sodium cation (Figure 1.9). The
electrostatic (ion–dipole) forces stabilize
17
this conformation, hampering the ring inversion up to 425 K. The
complexity of the r.t. 1 H NMR
spectrum recorded for the cyclic 33, demonstrated the slow exchange
of multiple conformations on the
NMR time scale. Stepwise addition of sodium picrate to 33, induced
the formation of a complex with a
remarkably simplified 1 H NMR spectrum. With an excess of guest, we
observed the formation of an 8-
fold symmetric species (Figure 1.10) was observed.
Figure 1.10. Picture of the predicted lowest energy conformations
for 33 without sodium cations.
Differently from the twenty-four-membered 33, the N-benzyloxyethyl
cyclic homologue 34 did not
yield any ordered conformation in the presence of cationic guests.
The association constants (Ka) for the
complexation of 32, 33 and 34 to the first group alkali metals and
ammonium, were evaluated in H2O–
CHCl3 following Cram‘s method (Table 1.1) 41
. The results presented in Table 1.1 show a good degree
of selectivity for the smaller cations.
Table 1.1 R, Ka, and G for cyclic peptoid hosts 32, 33 and 34
complexing picrate salt guests in CHCl3 at 25
C; figures within ±10% in multiple experiments, guest/host
stoichiometry for extractions was assumed as 1:1.
41 K. E. Koenig, G. M. Lein, P. Stuckler, T. Kaneda and D. J. Cram,
J. Am. Chem. Soc., 1979, 101, 3553.
18
The ability of cyclic peptoids to extract cations from bulk water
to an organic phase prompted us to
verify their transport properties across a phospholipid
membrane.
The two processes were clearly correlated although the latter is
more complex implying, after
complexation and diffusion across the membrane, a decomplexation
step. 42-43
In the presence of NaCl as
added salt, only compound 32 showed ionophoric activity while the
other cyclopeptoids are almost
inactive. Cyclic peptoids have different cation binding preferences
and, consequently, they may exert
selective cation transport. These results are the first indication
that cyclic peptoids can represent new
motifs on which to base artificial ionophoric antibiotics.
1.4.5 Catalytic Peptoids
An interesting example of the imaginative use of reactive
heterocycles in the peptoid field, can be
found in the foldamers mimics. Foldamers mimics are synthetic
oligomers displaying
conformational ordering. Peptoids have never been explored as
platform for asymmetric catalysis.
Kirshenbaum
reported the synthesis of a library of helical peptoid oligomers
enabling the oxidative
kinetic resolution (OKR) of 1-phenylethanol induced by the catalyst
TEMPO (2,2,6,6-
tetramethylpiperidine-1-oxyl) (figure 1.14) 44
Figure 1.14. Oxidative kinetic resolution of enantiomeric
phenylethanols 35 and 36.
The TEMPO residue was covalently integrated in properly designed
chiral peptoid backbones, which
were used as asymmetric components in the oxidative
resolution.
The study demonstrated that the enantioselectivity of the catalytic
peptoids (built using the chiral (S)-
and (R)-phenylethyl amines) depended on three factors: 1) the
handedness of the asymmetric
environment derived from the helical scaffold, 2) the position of
the catalytic centre along the peptoid
backbone, and 3) the degree of conformational ordering of the
peptoid scaffold. The highest activity in
the OKR (e.e. > 99%) was observed for the catalytic peptoids
with the TEMPO group linked at the N-
terminus, as evidenced in the peptoid backbones 39 (39 is also
mentioned in figure 1.14) and 40
(reported in figure 1.15). These results revealed that the
selectivity of the OKR was governed by the
global structure of the catalyst and not solely from the local
chirality at sites neighboring the catalytic
centre.
42 R. Ditchfield, J. Chem. Phys., 1972, 56, 5688. 43 K. Wolinski,
J. F. Hinton and P. Pulay, J. Am. Chem. Soc., 1990, 112, 8251. 44
G. Maayan, M. D. Ward, and K. Kirshenbaum, Proc. Natl. Acad. Sci.
USA, 2009, 106, 13679.
19
1.4.6 PNA and Peptoids Tagged With Nucleobases.
Nature has selected nucleic acids for storage (DNA primarily) and
transfer of genetic information
(RNA) in living cells, whereas proteins fulfill the role of
carrying out the instructions stored in the genes
in the form of enzymes in metabolism and structural scaffolds of
the cells. However, no examples of
protein as carriers of genetic information have yet been
identified.
Self-recognition by nucleic acids is a fundamental process of life.
Although, in nature, proteins are
not carriers of genetic information, pseudo peptides bearing
nucleobases, denominate peptide nucleic
acids (PNA, 41, figure 1.16), 4 can mimic the biological functions
of DNA and RNA (42 and 43, figure
1.16).
Figure 1.16. Chemical structure of PNA (19), DNA (20), RNA (21). B
= nucleobase
The development of the aminoethylglycine polyamide (peptide)
backbone oligomer with pendant
nucleobases linked to the glycine nitrogen via an acetyl bridge now
often referred to PNA, was inspired
by triple helix targeting of duplex DNA in an effort to combine the
recognition power of nucleobases
with the versatility and chemical flexibility of peptide chemistry
4 . PNAs were extremely good structural
mimics of nucleic acids with a range of interesting
properties:
DNA recognition,
Drug discovery:
Nanotechnology,
Pre-RNA world.
The very simple PNA platform has inspired many chemists to explore
analogs and derivatives in
order to understand and/or improve the properties of this class DNA
mimics. As the PNA backbone is
more flexible (has more degrees of freedom) than the phosphodiester
ribose backbone, one could hope
that adequate restriction of flexibility would yield higher
affinity PNA derivates.
The success of PNAs made it clear that oligonucleotide analogues
could be obtained with drastic
changes from the natural model, provided that some important
structural features were preserved.
The PNA scaffold has served as a model for the design of new
compounds able to perform DNA
recognition. One important aspect of this type of research is that
the design of new molecules and the
study of their performances are strictly interconnected, inducing
organic chemists to collaborate with
biologists, physicians and biophysicists.
An interesting property of PNAs, which is useful in biological
applications, is their stability to both
nucleases and peptidases, since the unnatural skeleton prevents
recognition by natural enzymes,
making them more persistent in biological fluids. 45
The PNA backbone, which is composed by repeating
N-(2 aminoethyl)glycine units, is constituted by six atoms for each
repeating unit and by a two atom
spacer between the backbone and the nucleobase, similarly to the
natural DNA. However, the PNA
skeleton is neutral, allowing the binding to complementary
polyanionic DNA to occur without repulsive
electrostatic interactions, which are present in the DNA:DNA
duplex. As a result, the thermal stability
of the PNA:DNA duplexes (measured by their melting temperature) is
higher than that of the natural
DNA:DNA double helix of the same length.
In DNA:DNA duplexes the two strands are always in an antiparallel
orientation (with the 5‘-end of
one strand opposed to the 3‘- end of the other), while PNA:DNA
adducts can be formed in two different
orientations, arbitrarily termed parallel and antiparallel (figure
1.17), both adducts being formed at room
temperature, with the antiparallel orientation showing higher
stability.
Figure 1.17. Parallel and antiparallel orientation of the PNA:DNA
duplexes
PNA can generate triplexes PAN-DNA-PNA, the base pairing in
triplexes occurs via Watson-Crick
and Hoogsteen hydrogen bonds (figure 1.18).
45 Demidov V.A., Potaman V.N., Frank-Kamenetskii M. D., Egholm M.,
Buchardt O., Sonnichsen S. H., Nielsen
P.E., Biochem. Pharmscol. 1994, 48, 1310.
21
Figure 1.18. Hydrogen bonding in triplex PNA2/DNA: C+GC (a) and TAT
(b)
In the case of triplex formation, the stability of these type of
structures is very high: if the target
sequence is present in a long dsDNA tract, the PNA can displace the
opposite strand by opening the
double helix in order to form a triplex with the other, thus
inducing the formation of a structure defined
as P-loop, in a process which has been defined as strand invasion
(figure 1.19). 46
Figure 1.19. Mechanism of strand invasion of double stranded DNA by
triplex formation
However, despite the excellent attributes, PNA has two serious
limitations: low water solubility 47
and
.
Many modifications of the basic PNA structure have been proposed in
order to improve their
performances in term of affinity and specificity towards
complementary oligonucleotide sequences. A
modification introduced in the PNA structure can improve its
properties generally in three different
ways:
ii) Improving sequence specificity, in particular for directional
preference (antiparallel vs parallel)
and mismatch recognition;
46 Egholm M., Buchardt O., Nielsen P.E., Berg R.H., J. Am. Chem.
Soc., 1992, 114,1895. 47 (a) U. Koppelhus and P. E. Nielsen, Adv.
Drug. Delivery Rev., 2003, 55, 267; (b) P. Wittung, J. Kajanus,
K.
Edwards, P. E. Nielsen, B. Nordén, and B. G. Malmstrom, FEBS Lett.,
1995, 365, 27. 48 (a) E. A. Englund, D. H. Appella, Angew. Chem.
Int. Ed., 2007, 46, 1414; (b) A. Dragulescu-Andrasi, S.
Rapireddy, G. He, B. Bhattacharya, J. J. Hyldig-Nielsen, G. Zon,
and D. H. Ly, J. Am. Chem. Soc., 2006, 128,
16104; (c) P. E. Nielsen, Q. Rev. Biophys., 2006, 39, 1; (d) A.
Abibi, E. Protozanova, V. V. Demidov, and M. D.
Frank-Kamenetskii, Biophys. J., 2004, 86, 3070.
22
Structure activity relationships showed that the original design
containing a 6-atom repeating unit
and a 2-atom spacer between backbone and the nucleobase was optimal
for DNA recognition.
Introduction of different functional groups with different
charges/polarity/flexibility have been
described and are extensively reviewed in several papers
49,50,51
. These studies showed that a constrained
flexibility was necessary to have good DNA binding (figure
1.20).
.
.
The shift
of the amide carbonyl groups away from the nucleobase (towards
thebackbone) and their replacement
with methylenes, resulted in a nucleosidated peptoid skeleton (44,
figure 1.21). Theoretical calculations
showed that the modification of the backbone had the effect of
abolishing the strong hydrogen bond
between the side chain carbonyl oxygen (α to the methylene carrying
the base) and the backbone amide
of the next residue, which was supposed to be present on the PNA
and considered essential for the
DNA hybridization.
Figure 1.21. Peptoid nucleic acid
49 a) Kumar, V. A., Eur. J. Org. Chem., 2002, 2021-2032. b)
Corradini R.; Sforza S.; Tedeschi T.; Marchelli R.;
Seminar in Organic Synthesis, Società Chimica Italiana, 2003,
41-70. 50 Sforza, S.; Haaima, G.; Marchelli, R.; Nielsen, P.E..
Eur. J. Org. Chem. 1999, 197-204. 51 Sforza, S.; Galaverna, G.;
Dossena, A.; Corradini, R.; Marchelli, R. Chirality, 2002, 14,
591-598. 52 O. Almarsson, T. C. Bruice, J. Kerr, and R. N.
Zuckermann, Proc. Natl. Acad. Sci. USA, 1993, 90, 7518.
23
.
work on the pairing properties of triazine heterocycles (as
recognition elements) linked to peptide and
peptoid oligomeric systems. In particular, when the backbone of the
oligomers was constituted by
condensation of iminodiacetic acid (45 and 46, Figure 1.22), the
hybridization experiments, conducted
with oligomer 45 and d(T) 12
, showed a T m
Figure 1.22. Triazine-tagged oligomeric sequences derived from an
iminodiacetic acid peptoid backbone.
This interesting result, apart from the implications in the field
of prebiotic chemistry, suggested the
preparation of a similar peptoid oligomers (made by iminodiacetic
acid) incorporating the classic
nucleobase thymine (47 and 48, figure 1.23) 54
.
Figure 1.23. Thymine-tagged oligomeric sequences derived from an
iminodiacetic acid backbone
The peptoid oligomers 47 and 48 showed thymine residues separated
by the backbone by the same
number of bonds found in nucleic acids (figure 1.24, bolded black
bonds). In addition, the spacing
between the recognition units on the peptoid framework was similar
to that present in the DNA (bolded
grey bonds).
Figure 1.24. Backbone thymines positioning in the peptoid oligomer
(47) and in the A-type DNA.
53 G. K. Mittapalli, R. R. Kondireddi, H. Xiong, O. Munoz, B. Han,
F. De Riccardis, R. Krishnamurthy, and A.
Eschenmoser, Angew. Chem. Int. Ed., 2007, 46, 2470. 54 R. Zarra, D.
Montesarchio, C. Coppola, G. Bifulco, S. Di Micco, I. Izzo, and F.
De Riccardis, Eur. J. Org.
Chem., 2009, 6113.
24
However, annealing experiments demonstrated that peptoid oligomers
47 and 48 do not hybridize
complementary strands of d(A) 16
or poly-r(A). It was claimed that possible explanations for those
results
resided in the conformational restrictions imposed by the charged
oligoglycine backbone and in the high
conformational freedom of the nucleobases (separated by two
methylenes from the backbone).
Small backbone variations may also have large and unpredictable
effects on the nucleosidated
peptoid conformation and on the binding to nucleic acids as
recently evidenced by Liu and co-
workers 55
,
with their synthesis and incorporation (in a PNA backbone) of
N-ε-aminoalkyl residues (49,
Figure 1.25).
N H
N N
49
O O O
Figure 1.25. Modification on the N - in an unaltered PNA
backbone
Modification on the γ-nitrogen preserves the achiral nature of PNA
and therefore causes no
stereochemistry complications synthetically.
Introducing such a side chain may also bring about some of the
beneficial effects observed of a
similar side chain extended from the R- or γ-C. In addition, the
functional headgroup could also serve as
a suitable anchor point to attach various structural moieties of
biophysical and biochemical interest.
Furthermore, given the ease in choosing the length of the peptoid
side chain and the nature of the
functional headgroup, the electrosteric effects of such a side
chain can be examined systematically.
Interestingly, they found that the length of the peptoid-like side
chain plays a critical role in determining
the hybridization affinity of the modified PNA. In the Liu
systematic study, it was found that short
polar side chains (protruding from the γ-nitrogen of peptoid-based
PNAs) negatively influence the
hybridization properties of modified PNAs, while longer polar side
chains positively modulate the
nucleic acids binding. The reported data did not clarify the reason
of this effect, but it was speculated
that factors different from electrostatic interaction are at play
in the hybridization.
1.5 Peptoid synthesis
The relative ease of peptoid synthesis has enabled their study for
a broad range of applications.
Peptoids are routinely synthesized on linker-derivatized solid
supports using the monomeric or
submonomer synthesis method. Monomeric method was developed by
Merrifield 2 and its synthetic
procedures commonly used for peptides, mainly are based on solid
phase methodologies (e.g. scheme
1.1).
The most common strategies used in peptide synthesis involve the
Boc and the Fmoc protecting
groups.
55 X.-W. Lu, Y. Zeng, and C.-F. Liu, Org. Lett., 2009, 11,
2329.
25
O
Peptoids can be constructed by coupling N-substituted glycines
using standard α-peptide synthesis
methods, but this requires the synthesis of individual monomers 4 ,
this is based by a two-step monomer
addition cycle. First, a protected monomer unit is coupled to a
terminus of the resin-bound growing
chain, and then the protecting group is removed to regenerate the
active terminus. Each side chain
requires a separate N α -protected monomer.
Peptoid oligomers can be thought of as condensation homopolymers of
N-substituted glycine. There
are several advantages to this method, but the extensive synthetic
effort required to prepare a suitable set
of chemically diverse monomers is a significant disadvantage of
this approach. Additionally, the
secondary N-terminal amine in peptoid oligomers is more sterically
hindered than primary amine of an
amino acid, for this reason coupling reactions are slower.
.
repeat Scheme 1.2. Sub-monomeric synthesis of peptoids
Sub-monomeric method consists in the construction of peptoid
monomer from C- to N-terminus
using N,N-diisopropylcarbodiimide (DIC)-mediated acylation with
bromoacetic acid, followed by
amination with a primary amine. This two-step sequence is repeated
iteratively to obtain the desired
oligomer. Thereafter, the oligomer is cleaved using trifluoroacetic
acid (TFA) or by
hexafluorisopropanol, scheme 1.2. Interestingly no protecting
groups are necessary for this procedure.
The availability of a wide variety of primary amines facilitates
the preparation of chemically and
structurally divergent peptoids.
1.6 Synthesis of PNA monomers and oligomers
The first step for the synthesis of PNA, is the building of PNA‘s
monomer. The monomeric unit is
constituted by an N-(2-aminoethyl)glycine protected at the terminal
amino group, which is essentially a
pseudopeptide with a reduced amide bond. The monomeric unit can be
synthesized following several
methods and synthetic routes, but the key steps is the coupling of
a modified nucleobase with the
secondary amino group of the backbone by using standard peptide
coupling reagents (N,N'-
dicyclohexylcarbodiimide, DCC, in the presence of
1-hydroxybenzotriazole, HOBt). Temporary
masking the carboxylic group as alkyl or allyl ester is also
necessary during the coupling reactions. The
56 R. N. Zuckermann, J. M. Kerr, B. H. Kent, and W. H. Moos, J. Am.
Chem. Soc., 1992, 114, 10646.
26
protected monomer is then selectively deprotected at the carboxyl
group to produce the monomer ready
for oligomerization. The choice of the protecting groups on the
amino group and on the nucleobases
depends on the strategy used for the oligomers synthesis. The
similarity of the PNA monomers with the
amino acids allows the synthesis of the PNA oligomer with the same
synthetic procedures commonly
used for peptides, mainly based on solid phase methodologies. The
most common strategies used in
peptide synthesis involve the Boc and the Fmoc protecting groups.
Some tactics, on the other hand,
are necessary in order to circumvent particularly difficult steps
during the synthesis (i.e. difficult
sequences, side reactions, epimerization, etc.). In scheme 1.3, a
general scheme for the synthesis of PNA
oligomers on solid-phase is described.
N H
N OH
O O
Scheme 1.3. Typical scheme for solid phase PNA synthesis.
The elongation takes place by deprotecting the N-terminus of the
anchored monomer and by
coupling the following N-protected monomer. Coupling reactions are
carried out with HBTU or, better,
its 7-aza analogue HATU 57
which gives rise to yields above 99%. Exocyclic amino groups
present on
cytosine, adenine and guanine may interfere with the synthesis and
therefore need to be protected with
semi-permanent groups orthogonal to the main N-terminal protecting
group.
In the Boc strategy the amino groups on nucleobases are protected
as benzyloxycarbonyl derivatives
(Cbz) and actually this protecting group combination is often
referred to as the Boc/Cbz strategy. The
Boc group is deprotected with trifluoroacetic acid (TFA) and the
final cleavage of PNA from the resin,
with simultaneous deprotection of exocyclic amino groups in the
nucleobases, is carried out with HF or
with a mixture of trifluoroacetic and trifluoromethanesulphonic
acids (TFA/TFMSA). In the Fmoc
strategy, the Fmoc protecting group is cleaved under mild basic
conditions with piperidine, and is
57 Nielsen P. E., Egholm M., Berg R. H., Buchardt O., Anti-Cancer
Drug Des. 1993, 8, 53.
27
therefore compatible with resin linkers, such as MBHA-Rink amide or
chlorotrityl groups, which can be
cleaved under less acidic conditions (TFA) or hexafluoisopropanol.
Commercial available Fmoc
monomers are currently protected on nucleobases with the
benzhydryloxycarbonyl (Bhoc) groups, also
easily removed by TFA. Both strategies, with the right set of
protecting group and the proper cleavage
condition, allow an optimal synthesis of different type of classic
PNA or modified PNA.
1.7 Aims of the work
The objective of this research, is to gain new insights in the use
of peptoids as tools for structural
studies and biological applications. Five are the themes developed
in the present thesis:
1. Carboxyalkyl Peptoid PNAs. N γ -carboxyalkyl modified peptide
nucleic acids (PNAs),
containing the four canonical nucleobases, were prepared via
solid-phase oligomerization. The inserted
modified peptoid monomers (figures 1.26: 50 and 51) were
constructed through simple synthetic
procedures, utilizing proper glycidol and iodoalkyl
electrophiles.
Figure 1.26. Modified peptoid monomers
Synthesis of PNA oligomers was realized by inserting modified
peptoid monomers into a canonical
PNA, by this way four different modified PNA oligomers were
obtained (figure 1.27).
Figure 1.27. Modified PNA.
Thermal denaturation studies performed, in collaboration with Prof.
R. Corradini from the University
of Parma, with complementary antiparallel DNA strands, demonstrated
that the length of the N γ -side
chain strongly influences the modified PNAs hybridization
properties. Moreover, multiple negative
GTAGAT*50CACT–Gly–NH2, 52
G T*50AGAT*50CAC T*50–Gly–NH2, 53
GTAGAT*51CACT–Gly–NH2, 54
G T*51AGAT*51CAC T*51–Gly–NH2, 55
N H
N N
28
charges on the oligoamide backbone, when present on γ-nitrogen C6
side chains, proved to be beneficial
for the oligomers water solubility and DNA hybridization
specificity.
2. Structural analysis of cyclopeptoids and their complexes. The
aim of this work was the
studies of structural properties of cyclopeptoids in their free and
complexed form (figure 1.27: 56, 57
and 58).
cyclohexapeptoid 58.
The synthesis of hexa- and tetra- N-benzyl glycine linear oligomers
and of hexa- N-metoxyethyl
glycine linear oligomer (59, 60 and 61, figure 1.28), was
accomplished on solid-phase (2-chlorotrityl
resin) using the sub-monomer approach. 58
HO N
n n
Figure 1.28. linear N-Benzyl-hexapeptoid 59, linear N-benzyl
tetrapeptoid 60 and linear N-
metoxyethyl-hexapeptoid 61.
58
R. N. Zuckermann, J. M. Kerr, B. H. Kent, and W. H. Moos, J. Am.
Chem. Soc., 1992, 114, 10646.
29
All cycles obtained were crystallized and caractherizated by X-ray
analysis in collaboration with
Dott. Consiglia Tedesco from the University of Salerno and Dott.
Loredana Erra from European
Synchrotron Radiation Facility (ESRF), Grenoble, France.
3. Cationic cyclopeptoids as potential macrocyclic nonviral
vectors.
The aim of this work was the synthesis of three different cationic
cyclopeptoids (figure 1.28: 62, 63
and 64) to assess their efficiency in DNA cell transfection, in
collaboration with Prof. G. Donofrio of
the University of Parma.
cyclohexapeptoid 64.
of carboxyethyl cyclopeptoids as possible contrast agents in
MRI. Three cyclopeptoids 65, 66 and 67 (figure 1.30) containing
polar side chains, were synthesized
and, in collaboration with Prof. S. Aime, of the University of
Torino, the complexation properties with
Gd 3+
were evaluated.
tetracarboxyethyl cyclopeptoids 67.
. In this work some linear and
cyclopeptoids with specific side chains (-SH groups) were
synthetized. The aim was to introduce, by
means of sulfur bridges, peptoid backbone constrictions and to
mimic natural defensins (figure 1.30,
block I: 68 hexa-linear and related cycles 69 and 70; block II: 71
octa-linear and related cycles 72 and
73; block III: 74 dodeca-linear and related cycles 75, 76 and 77;
block IV: 78 dodeca-linear diprolinate
and related cycles 79, 80 and 81).
HO
O
NHBoc
S
BocHN
S
70
Figure 1.30, block I. Structures of the hexameric linear (68) and
corresponding cyclic 69 and 70.
59
a) W. Wang, S.M. Owen, D. L. Rudolph, A. M. Cole, T. Hong, A. J.
Waring, R. B. Lal, and R. I.
Lehrer The Journal of Immunology, 2010, 515-520; b) D. Yang, A.
Biragyn, D. M. Hoover, J.
Lubkowski, J. J. Oppenheim Annu. Rev. Immunol. 2004, 181-215.
31
N
N
N
O
O
O
O
STr
71
N
O
O
NH
STr
HO
Figure 1.30, block II. Structures of octameric linear (71) and
corresponding cyclic 72 and 73
OH NN
NO
S
S
75
76
OH
NN
N
N
N
N
OO
O
O
O
O
S
NO
O
N
N
N
N
NH
O
O
O
O
S
77
Figure 1.30, block III. Structures of linear (74) and corresponding
cyclic 75, 76 and 77.
32
HO
81
Figure 1.30, block IV. Structures of dodecameric linear diprolinate
(78) and corresponding cyclic
79, 80 and 81.
2.1 Introduction
appreciable chemical simplicity, make PNA an invaluable tool in
molecular biology. 60
Unfortunately,
despite the remarkable properties, PNA has two serious limitations:
low water solubility 61
and poor
.
Considerable efforts have been made to circumvent these drawbacks,
and a conspicuous number of
new analogs have been proposed 63
, including those with the γ-nitrogen modified
N-(2-aminoethyl)-
glycine (aeg) units 64
an accurate investigation on the N γ -
methylated PNA hybridization properties was reported. In this study
it was found that the formation of
PNA-DNA (or RNA) duplexes was not altered in case of a 30% N γ
-methyl nucleobase substitution.
However, the hybridization efficiency per N-methyl unit in a PNA,
decreased with the increasing of the
N-methyl content.
The negative impact of the γ-N alteration reported by Nielsen, did
not discouraged further
investigations. The potentially informational triazine-tagged
oligoglycines systems, 66
the oligomeric
5a constitute convincing
example of γ-nitrogen beneficial modification. In particular, the
Liu group contribution, 5a
revealed an
unexpected electrosteric effect played by the N γ -side chain
length. In their stringent analysis it was
demonstrated that while short ω-amino N γ -side chains negatively
influenced the modified PNAs
hybridisation properties, longer ω-amino N γ -side chains
positively modulated nucleic acids binding. It
was also found that suppression of the positive ω-aminoalkyl charge
(i.e. through acetylation) caused no
.
, and
on the basis of poor hybridization properties showed by two fully
peptoidic homopyrimidine oligomers
synthesized by our group, 5b
it was decided to explore the effects of anionic residues at the
γ-nitrogen in
a PNA framework on the in vitro hybridization properties.
60 (a) Nielsen, P. E. Mol. Biotechnol. 2004, 26, 233-248; (b)
Brandt, O.; Hoheisel, J. D. Trends Biotechnol. 2004,
22, 617-622; (c) Ray, A.; Nordén, B. FASEB J. 2000, 14, 1041-1060.
61 Vernille, J. P.; Kovell, L. C.; Schneider, J. W. Bioconjugate
Chem. 2004, 15, 1314-1321. 62 (a) Koppelhus, U.; Nielsen, P. E.
Adv. Drug. Delivery Rev. 2003, 55, 267-280; (b) Wittung, P.;
Kajanus, J.;
Edwards, K.; Nielsen, P. E.; Nordén, B.; Malmstrom, B. G. FEBS
Lett. 1995, 365, 27-29. 63 (a) De Koning, M. C.; Petersen, L.;
Weterings, J. J.; Overhand, M.; van der Marel, G. A.; Filippov, D.
V.
Tetrahedron 2006, 62, 3248–3258; (b) Murata, A.; Wada, T. Bioorg.
Med. Chem. Lett. 2006, 16, 2933–2936; (c)
Ma, L.-J.; Zhang, G.-L.; Chen, S.-Y.; Wu, B.; You J.-S.; Xia, C.-Q.
J. Pept. Sci. 2005, 11, 812–817. 64 (a) Lu, X.-W.; Zeng, Y.; Liu,
C.-F. Org. Lett. 2009, 11, 2329-2332; (b) Zarra, R. ; Montesarchio,
D.; Coppola,
C.; Bifulco, G.; Di Micco, S.; Izzo, I.; De Riccardis, F. Eur. J.
Org. Chem. 2009, 6113-6120; (c) Wu, Y.; Xu, J.-C.;
Liu, J.; Jin, Y.-X. Tetrahedron 2001, 57, 3373-3381; (d) Y. Wu,
J.-C. Xu. Chin. Chem. Lett. 2000, 11, 771-774. 65 Haaima, G.;
Rasmussen, H.; Schmidt, G.; Jensen, D. K.; Sandholm Kastrup, J.;
Wittung Stafshede, P.; Nordén, B.;
Buchardt, O.; Nielsen, P. E. New. J. Chem. 1999, 23, 833-840. 66
Mittapalli, G. K.; Kondireddi, R. R.; Xiong, H.; Munoz, O.; Han,
B.; De Riccardis, F.; Krishnamurthy, R.;
Eschenmoser, A. Angew. Chem. Int. Ed. 2007, 46, 2470-2477. 67 The
authors suggested that longer side chains could stabilize amide Z
configuration, which is known to have a
stabilizing effect on the PNA/DNA duplex. See: Eriksson, M.;
Nielsen, P. E. Nat. Struct. Biol., 1996, 3, 410-413.
34
The N-(carboxymethyl) and the N-(carboxypentamethylene) N γ
-residues, present in the monomers 50
and 51 (figure 2.1) were chosen in order to evaluate possible side
chains length-dependent thermal
denaturations effects, and with the aim to respond to the pressing
water-solubility issue, which is crucial
for the specific subcellular distribution 68
.
The synthesis of a negative charged
N-(2-carboxyalkylaminoethyl)-glycine backbone (negative
charged PNA are rarely found in literature) 69
was based on the idea to take advantage of the availability
of a multitude of efficient methods for the gene cellular delivery
based on the interaction of carriers with
negatively charged groups. Most of the nonviral gene delivery
systems are, in fact, based on cationic
lipids 70
interacting with negative charged genetic vectors. Furthermore,
the
neutral backbone of PNA prevents them to be recognized by proteins
which interact with DNA, and
PNA-DNA chimeras should be synthesized for applications such as
transcription factors scavenging
(decoy) 72
or activation of RNA degradation by RNase-H (as in antisense
drugs).
This lack of recognition is partly due to the lack of negatively
charged groups and of the
corresponding electrostatic interactions with the protein
counterpart 73
.
In the present work we report the synthesis of the bis-protected
thyminylated N γ -ω-carboxyalkyl
monomers 50 and 51 (figure 2.1), the solid-phase oligomerization
and the base-pairing behaviour of
four oligomeric peptoid sequences 52-55 (figure 2.2) incorporating,
in various extent and in different
positions, the monomers 50 and 51.
68 Koppelius, U.; Nielsen, P. E. Adv. Drug Deliv. Rev. 2003, 55,
267-280. 69 (a) Efimov, V. A.; Choob, M. V.; Buryakova, A. A.;
Phelan, D.; Chakhmakhcheva, O. G. Nucleosides,
Nucleotides Nucleic Acids 2001, 20, 419-428; (b) Efimov, V. A.;
Choob, M. V.; Buryakova, A. A.; Kalinkina, A.
L.; Chakhmakhcheva, O. G. Nucleic Acids Res. 1998, 26, 566-575; (c)
Efimov, V. A.; Choob, M. V.; Buryakova,
A. A.; Chakhmakhcheva, O. G. Nucleosides Nucleotides 1998, 17,
1671-1679; (d) Uhlmann, E.; Will, D. W.;
Breipohl, G.; Peyman, A.; Langner, D.; Knolle, J.; O‘Malley, G.
Nucleosides Nucleotides 1997, 16, 603-608; (e)
Peyman, A.; Uhlmann, E.; Wagner, K.; Augustin, S.; Breipohl, G.;
Will, D. W.; Schäfer, A.; Wallmeier, H. Angew.
Chem. Int. Ed. 1996, 35, 2636–2638. 70 Ledley, F. D. Hum. Gene
Ther. 1995, 6, 1129–1144. 71 Wu, G. Y.; Wu, C. H. J. Biol. Chem.
1987, 262, 4429–4432. 72Gambari, R.; Borgatti, M.; Bezzerri, V.;
Nicolis, E.; Lampronti, I.; Dechecchi, M. C.; Mancini, I.;
Tamanini, A.;
Cabrini, G. Biochem. Pharmacol., 2010, 80, 1887-1894. 73 Romanelli,
A.; Pedone, C.; Saviano, M.; Bianchi, N.; Borgatti, M.; Mischiati,
C.; Gambari R. Eur. J. Biochem.,
2001, 268, 6066–6075.
35
Figure 2.2. Structures of target oligomers 52-55. T* represents the
modified thyminylated N γ -ω-
carboxyalkyl monomers. T*50 incorporates monomer 50, T*51
incorporates monomer 51.
The carboxy termini of the modified mixed purine/pyrimidine decamer
PNA sequences were linked
to a glycinamide unit. T*1 and T*2 represent the insertion of the
modified 50 and 51 N γ -ω-carboxyalkyl
monomer units, respectively.
The mixed-base sequence has been chosen since it has been proposed
by Nielsen and coworkers and
subsequently used by several groups as a benchmark for the
evaluation of the effect of modification of
the PNA structure on PNA:DNA thermal stability 74
.
2.2.1 Chemistry
The elaboration of monomers 50 and 51 (figure 2.1), suitable for
the Fmoc-based oligomerization,
took advantage of the chemistry utilized to construct the regular
PNA monomers. In particular, the
synthesis of the N-protected monomer 50 started with the
t-Bu-glycine (82) glycidol amination 5 , as
shown in scheme 2.1. N-fluorenylmethoxycarbonyl protection of the
adduct 85, and subsequent diol
oxidative cleavage, gave the labile aldehyde 86. Compound 86 was
subjected to reductive amination in
the presence of methylglycine to obtain the triply protected
bis-carboxyalkyl ethylenediamine key
intermediate 87.
The 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU),
promoted condensation of 87 with thymine-1-acetic acid gave the
expected tertiary amide 88.
Careful LiOH-mediated hydrolysis allowed to preserve the
base-labile Fmoc group, affording the
target monomer unit 50.
74 (a) Sforza, S.; Tedeschi, T.; Corradini, R.; Marchelli, R. Eur.
J. Org. Chem., 2007, 5879–5885; (b) Englund, E.
A.; Appella, D. H. Org. Lett., 2005, 7, 3465-3467; (c) Sforza, S.;
Corradini, R.; Ghirardi, S.; Dossena, A.;
Marchelli, R. Eur. J. Org. Chem., 2000, 2905-2913.
GTAGAT*50CACT–Gly–NH2, 52
G T*50AGAT*50CAC T*50–Gly–NH2, 53
GTAGAT*51CACT–Gly–NH2, 54
G T*51AGAT*51CAC T*51–Gly–NH2, 55
36
Scheme 2.1. Synthesis of the PNA monomer 50. Reagents and
conditions: a) glycidol, DMF,
DIPEA, 70°C, 3 days, 41%; b) fluorenylmethoxycarbonyl chloride
(Fmoc-Cl), NaHCO3, 1,4-
dioxane/H2O, overnight, 63%; c) NaIO4, THF/H2O, 2h, 97%; d)
H2NCH2COOCH3, NaHB(AcO)3,
triethylamine in CH2Cl2, overnight, 70%; e) thymine-1-acetic acid,
Et3N, HATU in DMF, overnight,
49%; f) LiOH . H2O, 1,4-dioxane /H2O, 0°C, 30 min., 69%.
The synthesis of compound 51 required a different strategy, due to
the low yields obtained in the
glycidol opening induced by the t-butyl ester of the 6-aminocaproic
acid (89, see the experimental
section). A better electrophile was devised in the benzyl
2-iodoethylcarbamate (65, 75
Scheme 2.2). The
nucleophilic displacement gave the secondary amine 95, containing
the Cbz-protected ethylendiamine
core. Compound 95, after a straightforward protective group
adjustment and a subsequent reductive
amination, produced the fully protected bis-carboxyalkyl
ethylenediamine key intermediate 98. This last
was reacted with thymine-1-acetic acid and
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP), as condensing agent, and gave the
amide 99. Finally, after careful
chemoselective hydrolysis of the methyl ester, the required monomer
51 was obtained in acceptable
yields.
75 Bolognese, A.; Fierro O.; Guarino, D.; Longobardo, L.; Caputo,
R. Eur. J. Org. Chem. 2006, 169-173.
O
85, R = Fmoc
f
37
Scheme 2.2. Synthesis of the PNA monomer 51. Reagents and
conditions: a) t-Butanol, DMAP, DCC, CH2Cl2,
overnight, 58%; b) H2, Pd/C (10 % w/w), acetic acid, methanol, 1h
and 30 min., quant.; c) Cbz-Cl, CH2Cl2, 0°C,
overnight, quant.; d) I2, imidazole, PPh3, CH2Cl2, 3h, 77%; e)
K2CO3, CH3CN, reflux, overnight, 67%, f)
fluorenylmethoxycarbonyl chloride (Fmoc-Cl), NaHCO3,
1,4-dioxane/H2O, overnight, 97%; g) H2, Pd/C (10 %
w/w), acetic acid, methanol; 1h, quant.; h) ethyl glyoxalate,
NaHB(AcO)3, triethylamine in CH2Cl2, overnight,
25%; i) thymine-1-acetic acid, Et3N, PyBOP in DMF; overnight, 70%
l) LiOH, 1,4-dioxane/H2O, 30 min., 30%.
The oligomers 52-55 were manually assembled in a stepwise fashion
on a Rink-amide NOVA-PEG
resin solid support. The unmodified PNA monomers were coupled using
2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluoro-phosphate (HBTU). HATU was
used for the coupling reactions
involving the less reactive secondary amino groups of the modified
monomers 50 and 51. The decamers
were detached from the solid support and quantitatively deprotected
from the t-butyl protecting groups,
using a 9:1 mixture of trifluoroacetic acid and m-cresol. The
water-soluble oligomers were purified by
RP-HPLC, yielding the desired 52-55 as pure compounds. Their
identity was confirmed by MALDI-
TOF mass spectrometry.
2.2.2 Hybridization studies
In order to verify the ability of decamers 49-52 to bind
complementary DNA, UV-monitored melting
experiments were performed mixing the water-soluble oligomers with
the complementary antiparallel
O
HO
NHCbz
89
5
O
t-BuO
NH2
5
a
96, R = Fmoc, R' = Cbz f
R
h
H N
38
deoxyribonucleic strands (5 μM concentration, ε= 260 nm). Table 2.1
presents the thermal stability
studies of the duplexes formed between the modified PNAs and the
DNA anti-parallel strand, in
comparison with the unmodified PNA.
The data obtained clearly demonstrated that the distance of the
negative charged carboxy group from
the oligoamide backbone strongly affects the PNA:DNA duplex
stability. In particular, when the γ-
nitrogen brings an acetic acid substituent (with a single methylene
distancing the oligoamide backbone
and the charged group, entry 2), a drop of 5.4 °C in Tm of the
carboxypeptoid-PNA/DNA(ap) duplex is
observed, when compared with unmodified PNA (entry 1). Triple
insertion of monomer 50 (entry 3),
results in a decrease of 2.6 °C per N-acetyl unit, showing no N γ
-substitution detrimental additive effects
on the annealing properties. In both cases the ability to
discriminate closely related sequences is
magnified, respect to the unmodified PNA.
Table 2.1. Thermal stabilities (Tm, °C) of modified PNA/DNA
duplexes
Entry PNA Anti-parallel DNA
3 GT*50AGAT*50CACT*50–Gly–NH2, (53) 40.7 34.4
4 GTAGAT*51CACT–Gly–NH2, (54) 44.8 30.8
5 G T*51AGAT*51CAC T*51–Gly–NH2, (55) 44.1 35.6
6 5‘–GTAGATCACT–3‘
a 5‘–AGTGATCTAC–3‘
b 5‘–AGTGGTCTAC–3‘
For the binding of the N γ -caproic acid derivatives with the
full-matched antiparallel DNA, the table
shows an evident increase of the affinity (entry 4 and 5), when
compared with the modified sequences
with shorter side chains (entry 2 and 3). Comparison with the
corresponding aegPNA showed, for the
single insertion, a 3.8 °C Tm drop, while, for triple substitution,
a Tm decrease of 1.5 °C per N γ -alkylated
monomer. It is also worth noting, in both 54 and 55, the slight
increase of the binding specificity (ΔTm =
5.6 °C and 0.8 °C, entry 4 and 5) respect to unmodified PNA.
In previous studies, reporting the performances of backbone
modified PNA containing negatively
charged monomers derived from amino acids, the drop in melting
temperature was found to be 3.3 °C in
the case of L-Asp monomer and 2.3° C in the case of D-Glu. The
present results are in line with these
data, with a decrease in melting temperatures which still allows
stronger binding than natural DNA
(entry 6). Thus it is possible to introduce negatively charged
groups via alkylation of the amide nitrogen
in the PNA backbone without significant loss of stability of the
PNA-DNA duplex, provided that a five
methylene spacer is used.
2.3. Conclusions
In this work, we have constructed two orthogonally protected N
--carboxy alkylated units. The
successful insertion in PNA-based decamers, through standard
solid-phase synthesis protocols, and the
following hybridization studies, in the presence of DNA
antiparallel strand, demonstrate that the N -
substitution with negative charged groups is compatible with the
formation of a stable PNA:DNA
duplex. The present study also extends the observation that
correlates the efficacy of the nucleic acids
hybridization with the length of the N alkyl substitution,
5a expanding the validity also to N
--negative
charged side chains. The newly produced structures can create new
possibilities for PNA with
functional groups enabling further improvement in their ability to
perform gene-regulation.
2.4 Experimental section
2.4.1 General Methods.
All reactions involving air or moisture sensitive reagents were
carried out under a dry argon or
nitrogen atmosphere using freshly distilled solvents.
Tetrahydrofuran (THF) was distilled from LiAlH4
under argon. Toluene and CH2Cl2 were distilled from CaH2. Glassware
was flame-dried (0.05 Torr)
prior to use. When necessary, compounds were dried in vacuo over
P2O5 or by azeotropic removal of
water with toluene under reduced pressure. Starting materials and
reagents purchased from commercial
suppliers were generally used without purification unless otherwise
mentioned. Reaction temperatures
were measured externally; reactions were monitored by TLC on Merck
silica gel plates (0.25 mm) and
visualized by UV light, I2, or by spraying with H2SO4-Ce(SO4)2,
phosphomolybdic acid or ninhydrin
solutions and drying. Flash chromatography was performed on Merck
silica gel 60 (particle size: 0.040-
0.063 mm) and the solvents employed were of analytical grade.
Yields refer to chromatographically and
spectroscopically ( 1 H- and
13 C-NMR) pure materials. The NMR spectra were recorded on Bruker
DRX
400, ( 1 H at 400.13 MHz,
13 C at 100.03 MHz), Bruker DRX 250 (
1 H at 250.13 MHz,
13 C at 62.89 MHz),
and Bruker DRX 300 ( 1 H at 300.10 MHz,
13 C at 75.50 MHz) spectrometers. Chemical shifts () are
reported in ppm relatively to the residual solvent peak (CHCl3, =
7.26, 13
CDCl3, : = 77.0; CD2HOD,
= 3.34, 13
CD3OD, = 49.0) and the multiplicity of each signal is designated by
the following
abbreviations: s, singlet; d, doublet; t, triplet; q, quartet;
quint, quintuplet; m, multiplet; br, broad.
Coupling costants (J) are quoted in Hz. Homonuclear decoupling,
COSY-45 and DEPT experiments
completed the full assignment of each signal. Elemental analyses
were performed on a CHNS-O
FlashEA apparatus (Thermo Electron Corporation) and are reported in
percent abundance. High
resolution ESI-MS spectra were performed on a Q-Star Applied
Biosystem mass spectrometer. ESI-MS
analysis in positive ion mode was performed using a Finnigan LCQ
Deca ion trap mass spectrometer
(ThermoFinnigan, San Josè, CA, USA) and the mass spectra were
acquired and processed using the
Xcalibur software provided by Thermo Finnigan. Samples were
dissolved in 1:1 CH3OH/H2O, 0.1 %
formic acid, and infused in the ESI source by using a syringe pump;
the flow rate was 5 μl/min. The
capillary voltage was set at 4.0 V, the spray voltage at 5 kV, and
the tube lens offset at -40 V. The
capillary temperature was 220 °C. MALDI TOF mass spectrometric
analyses were performed on a
PerSeptive Biosystems Voyager-De Pro MALDI mass spectrometer in the
Linear mode using -cyano-
4-hydroxycinnamic acid as the matrix. HPLC analyses were performed
on a Jasco BS 997-01 series,
equipped with a quaternary pumps Jasco PU-2089 Plus, and an UV
detector Jasco MD-2010 Plus. The
40
125Å, 7.8 × 300 mm).
Tert-butyl 2-(2,3-dihydroxypropylamino)acetate (55).
To a solution of glycidol (83, 436 μL, 6.56 mmol) in DMF (5 mL),
glycine t-butyl ester (82, 1.00 g,
5.96 mmol) in DMF (10 mL), and DIPEA (1600 μL, 8.94 mmol) were
added. The reaction mixture was
refluxed for three days. NaHCO3 (0.50 g, 5.96 mmol) was added and
the solvent was concentrated in
vacuo to give the crude product, which was purified by flash
chromatography (CH2Cl2/CH3OH/NH3 2.0
M solution in ethyl alcohol, from: 100/0/0.1 to 88/12/0.1) to give
84 (0.50 g, 41%) as a yellow pale oil;
[Found: C, 52.7; H, 9.4. C9H19NO4 requires C, 52.67; H, 9.33%]; Rf
(97/3/0.1, CH2Cl2/CH3OH/NH3
2.0M solution in ethyl alcohol) 0.36; H (400.13 MHz CDC13) 1.42
(9H, s, (CH3)3C), 2.62 (1H, dd, J
12.0, 7.7 Hz, CHHCH(OH)CH2OH), 2.71 (1H, dd, J 12.0, 2.9 Hz,
CHHCH(OH)CH2OH), 3.28 (2H, br
s, CH2COOt-Bu), 3.51 (1H, dd, J 11.0, 5.4 Hz, CH2CH(OH)CHHOH), 3.62
(1H, dd, J 11.0, 1.2 Hz,
CH2CH(OH)CHHOH), 3.72 (1H, m, CH2CH(OH)CH2OH); C (100.03 MHz,
CDCl3) 29.2, 52.6, 53.1,
66.4, 71.6, 82.7, 172.8; m/z (ES) 206 (MH + ); (HRES) MH
+ , found 206.1390. C9H20NO4
(9H-fluoren-9-yl) methyl (tert-butoxycarbonyl) methyl
2,3dihydroxypropylcarbamate (85):
To a solution of 84 (0.681 g, 3.33 mmol) in a 1:1 mixture of
1,4-dioxane/water (46 mL), NaHCO3
(0.559 g, 6.66 mmol) was added. The mixture was sonicated until
complete dissolution and, Fmoc-Cl
(1.03 g, 3.99 mmol) was added. The reaction mixture was stirred
overnight, then, through addition of a
saturated solution of NaHSO4, the pH was adjusted to 3 and the
solvent was concentrated in vacuo to
remove the excess of 1,4-dioxane. The water layer was extracted
with CH2Cl2 (three times), the organic
phase was dried over MgSO4, filtered and the solvent evaporated in
vacuo to give the crude product,
which was purified by flash chromatography (CH2Cl2/CH3OH, from:
100/0 to 90/10) to give 85 (0.90 g,
63%) as a yellow pale oil; [Found: C, 67.4; H, 6.9. C24H29NO6
requires C, 67.43; H, 6.84%]; Rf
(95/5/0.1, CH2Cl2/CH3OH/NH3 2.0M solution in ethyl alcohol) 0.44; H
(300.10 MHz CDC13, mixture
of rotamers) 1.45 (9H, s, (CH3)3C), 3.04-3.25 (1.7 H, m,
CH2CH(OH)CH2OH), 3.40 (0.3 H, m,
CH2CH(OH)CH2OH), 3.43-3.92 (3H, m, CH2CH(OH)CH2OH, CH2CH(OH)CH2OH),
3.93 (2H, br s,
CH2COOt-Bu), 4.22 (0.9H, m, CH-Fmoc and CH2-Fmoc), 4.42 (1.4H, br
d. J 9.0 Hz, CH2-Fmoc), 4.61
(0.7H, m, J 9.0 Hz, CH-Fmoc), 7.29 (2H, br t, J 7.0 Hz, Ar.
(Fmoc)), 7.38 (2H, br t, J 7.0 Hz, Ar.
(Fmoc)), 7.57 (2H, br d, J 9.0 Hz, Ar. (Fmoc)), 7.76 (2H, br d, J
9.0 Hz, Ar. (Fmoc); C (75.50 MHz,
CDCl3, mixture of rotamers) 28.2, 47.4, 52.1, 52.3, 52.9, 53.2,
63.5, 64.0, 67.3, 68.1, 68.5, 70.1, 70.5,
83.1, 120.1, 120.2, 124.9, 125.2, 127.3, 127.9, 128.0, 141.5,
143.8, 156.4, 157.3, 170.4, 171.3; m/z (ES)
428 (MH + ); (HRES) MH
(9H-fluoren-9-yl) methyl (tert-butoxycarbonyl)
methylformylmethylcarbamate (86):
To a solution of 85 (0.80 g, 1.87 mmol) in a 5:1 mixture of THF and
water (5 mL), sodium periodate
(0.44 g, 2.06 mmol) was added in one portion. The mixture was
sonicated for 15 min and stirred for
another 2 hours at room temperature. The reaction mixture was
filtered, the filtrate was washed with
CH2Cl2 and the solvent evaporated in vacuo. The crude product was
dissolved in CH2Cl2/H2O, and the
organic phase was dried over MgSO4, filtered and the solvent
evaporated in vacuo to